The Microsporidia are zoonotic pathogenic protists which can be transmitted by food or water. They are classified as category B priorty pathogens by the NIH and are also on the EPA high priority organism and contaminant lists. The most common human pathogenic microsporidia Enterocytozoon bieneusi is non cultivatable; however other pathogenic microsporidia such as Encephalitozoon cuniculi and Encephalitozoon hellem can be grown in vitro and in addition small animal models of Encephalitozoonidae infections exist allowing detailed pathogenesis studies in the laboratory. What is lacking for the characteriziation of these enigmatic protists are techniques which would permit the genetic manipulation of these organisms providng the tools for disseciton of the biology of these organisms. We have studied and characterized microsporidian methionine aminopeptidase type 2 (MetAP2) which is an essential enzyme in these organisms. MetAP2 is a logical therapeutic target as microsporidia lack MetAP1 making MetAP2 an essential enzyme. Using human MetAP2 data we have been able to modify microsporidian MetAP2 to make it resistant to ovalicin, a known inhibitor of MetAP2. This provides the first selectable marker for genetic studies in these organisms. We propose to build on this work to develop a useful transfection system for the microsporidia using E. cunicuili and E. hellem. This application will also take advantage of the experience of our collaborators in the development of transfection systems for various Apicomplexa and our characterization of the E. cuniculi and E. hellem genomic rRNA loci. The development of microsporidian transfection, while high risk, would have a major impact on the field and would solve a major research impediment for study of these emerging pathogens. PUBLIC HEALTH RELEVANCE: Microsporidia cause opportunistic infections in patients with AIDS and are emerging infections in immune competent patients. Research on these enigmatic pathogens has been limited by the absence of genetic systems that would allow examination of gene function. We propose to develop such genetic systems based on our previous laboratory work that resulted in the identification of the first potential selectable marker for these organisms.